The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the alpha- and beta-globin gene loci in an erythroid cell line

Abstract

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human beta-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous beta- or alpha-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Figure 1

A functional TATA box and the core NF-E2 sites are required for enhancer-dependent transcriptional activation. (Upper) The constructs used to examine the interaction between the HS2 enhancer and minimal γ-globin promoter. The open box represents the −356+35 minimal γ- globin promoter. The hatched box represents the luciferase reporter gene. The stippled box represents the 1,900-bp KpnI–BglII fragment of HS2 of the β-globin LCR. The NF-E2 motifs are approximately 1 kb upstream of the TATA box. Constructs 3 and 4 contain mutated TATA box and NF-E2 binding motifs, respectively. (Lower) The fold activation by the enhancer relative to expression of construct 1.

Figure 2

Human NF-E2 interacts specifically with dTAF_II110. (A) The yeast two-hybrid assay demonstrates that p45NF-E2 interacts with dTAF_II110. The Saccharomyces cerevisiae reporter strain HF7C was transformed with the indicated plasmids. AD-NF-E2 contains the entire coding sequence of human NF-E2 inserted in-frame with the activation domain of GAL4 (amino acids 768–881). DB-TBP, DB-TAF_II55, DB-TAF_II70, and DB-TAF_II250 contain the coding sequence of the human proteins and DB-TAF_II110 the coding sequence of the Drosophila protein inserted in-frame to the GAL4 DNA binding domain (amino acids 1–147). A specific interaction between DB-p53 and AD-simian virus 40 T antigen has been reported previously. Leu- and Trp- transformants were streaked onto synthetic medium plates lacking tryptophan, leucine, and histidine to assess potential interactions (Right) and synthetic medium plates lacking tryptophan and leucine to confirm plating efficiency (Middle). The plates were incubated at 30°C for 3 days. (B) The yeast two-hybrid assay demonstrates that the NF-E2/dTAF_II110 interaction is specific. DB-TAF110 was plated in combination with the simian virus 40 T antigen or GAL4-AD. AD-NF-E2 was plated in combination with TAF_II110, TAF_II70, or the GAL4-DB. Transfections were grown in the absence of leucine and tryptophan to assess transformation efficiency (Middle) or in the absence of leucine, tryptophan and histidine to assess protein-protein interactions (Right). Cotransfection of AD-simian virus 40 T-antigen and DB-p53 served as the positive control. (C) GST chromatography confirms that dTAF_II110 binds specifically to p45NF-E2. One microgram of GST or GST-NF-E2, bound to glutathione-Sepharose was incubated with in vitro-translated dTAF_II110 as described in Materials and Methods. Bound proteins were detected by SDS/PAGE followed by autoradiography. An example of labeled dTAF_II110 that was loaded is shown in lane L.

Figure 3

Colocalization of the activation domain of NF-E2 with the TAF_II130 binding domain. (A) Localization of activation domain of NF-E2. K562 cells were cotransfected by electroporation with expression vectors for GAL4 fusion proteins containing the indicated regions of NF-E2 and a reporter construct containing five GAL4 DNA binding sites regulating the expression of the chloramphenicol acetyltransferase gene (pG5EC). The first 312 amino acids of NF-E2 previously have been shown to contain a proline-rich activation domain lacking structural homology to other proteins denoted by a line, and a basic region similar to other cap’n-collar transcriptional activators denoted by an open box. The expression vector used in this study was pCINeo, containing the cytomegalovirus promoter. The fold activation represents the chloramphenicol acetyltransferase activity relative to the expression of the expression vector containing the GAL4 DNA binding domain alone. (B) hTAF_II130 interacts with the NF-E2 activation domain in the yeast two-hybrid assay. The S. cerevisiae reporter strain SFY526, which contains a β-galactosidase reporter construct under the control of a GAL4 binding motif multimer, was transformed with the indicated plasmids. The AD-NF-E2 constructs contain the stated regions of the coding sequence of human NF-E2 inserted in-frame to the activation domain of GAL4 (amino acids 768–881). DB-TAF_II130 contains the partial coding sequence of the human protein inserted in-frame to the GAL4 DNA binding domain (amino acids 1–147). (C) The amino terminal 80 amino acids of NF-E2 interacts specifically with hTAF_II130 in the GST chromatography system. Fusion proteins containing GST fused to amino acids 1–374, 1–80, or 80–374 of NF-E2 or GST alone were expressed in Escherichia coli and bound to glutathione-Sepharose beads. The beads then were incubated with ³⁵S-labeled in vitro-translated hTAF_II130. Other experimental details are as stated in Fig. 2C.

Figure 4

The TAF_II130 binding domain of NF-E2 is essential for enhancer-dependent activation of the β-globin gene. (A) Wild-type NF-E2 and NF-E2Δ80 expression in selected CB3 clones as demonstrated in a gel mobility shift assay. CB3 cells were stably transfected with pCINeoHA vector alone or pCINeoHA vectors in which the human NF-E2 cDNA or NF-E2Δ80 was fused in-frame with the HA tag. Nuclear extract was prepared from individual clones and analyzed by gel mobility shift assay, dimethyl sulfoxide-induced MEL extract serving as a control. ³²P-labeled DNA fragments containing the tandem NF-E2 binding sites in HS2 were incubated with individual extracts and electrophoresed on a 4% polyacrylamide gel. Lanes 1 and 2 show MEL cell and CB3 control extracts, the latter lacking the characteristic NF-E2 complex. Lane 3 shows a representative CB3 clone transfected with pCINeoHANF-E2 demonstrating a complex that comigrates with the NF-E2 complex observed in MEL cells. Lane 4 verified the presence of HA-tagged NF-E2 with specific ablation of the NF-E2 complex by the addition of anti-HA antibody (anti-HA). Lanes 5 and 6 show similar results for a clone expressing pCINeoHANF-E2Δ80. (B) High-level expression of a transiently transfected β-globin promoter containing construct is dependent on wild-type NF-E2 expression in CB3 cells. CB3 clones expressing wild-type NF-E2 or NF-E2Δ80 or CB3 cells transfected with the pCINeo vector (control) were transiently transfected with a plasmid containing HS2 linked to a β-promoter/luciferase gene hybrid. Whole cell extracts were assayed for luciferase activity. The results shown are representative of the three transfection experiments. MEL cells were transfected as a positive control. The fold activation observed was calculated relative to the expression of the HS2βLuciferase construct in CB3 cells.

Figure 5

High-level endogenous globin gene expression is observed only with wild-type p45NF-E2 expression in erythroid cells. RNase protection assays for α-globin, β-major mRNAs from CB3 cells, CB3 cells stably transfected with various p45 NF-E2 expressing constructs, and MEL cells. Actin levels were measured in parallel in all samples.